1. Technical Field
This invention relates to a solvent delivery pump assembly which is used for high performance liquid chromatography (hereinafter called HPLC) systems. It removes air bubbles and gaseous components dissolved in eluent (solvent) which is separated and analyzed by the liquid chromatography, thereby allowing to make a fine and precise solvent delivery at a high speed required for HPLC.
2. Background Art
There is a tendency that HPLC used to separate components in a given sample is made more and more highly accurate. Usually in this type of HPLC, an eluent (solvent) drawn from a reservoir by a solvent delivery pump is delivered via a sample injection valve to a detecting section including a separation column. Detected signals are recorded or sent to a monitor screen. In high speed and high accuracy liquid chromatography systems (known as semi-micro HPLC and micro HPLC) which require high accuracy in delivering the eluent under a high pressure yet at a very small quantity, it is common to install such phase separator (gas/liquid separator) as an air trap or a degasser (degassing unit) on the inlet side of the solvent delivery pump in order to insure the stability of the pump.
The purpose of this type of degassing unit is to remove unnecessary gases (air and other gases) dissolved in the eluent. For example, when an electrode reduction reaction is measured, any oxygen dissolved in the eluent greatly influences its measured value. That is, the reduction reaction of the oxygen itself causes a big background current, thereby causing noises to be increased.
FIG. 5 is a block diagram of a system configuration of HPLC. An eluent 2 in a first reservoir 1 is drawn up by a pump 5 through a pipe 3 and degassed by a phase separator 4. It is then delivered through a sample injection valve (auto sampler) 6 and a column 7 to a detector unit 8. The eluent delivered from the detector unit 8 flows out to a second reservoir 10 as a waste eluent 9. The reference arrow marks show the direction of the eluent delivery. Data detected by the detector unit 8 are transferred to a data processing unit 11, wherein they are processed in a visual form or a computer processable data form to provide and store. The column 7 is accommodated in an isothermal oven 7A to prevent the influence of external temperature. The pump 5 and the sample injection valve 6 are controlled by a system controller 12. The phase separator 4 is installed before the pump 5 to insure the stable delivery of the eluent and the accurate analysis by removing gases dissolved in the eluent which is drawn up from the first reservoir 1 by the pump 5.
As the other units and components consisting of this kind of high accuracy liquid chromatography system as well as the function of the whole system are well known so those explanations are omitted.
When the area of eluent delivery rate shifts from the range of 0.01-1.5 ml/min. for conventional HPLC systems to that of 1-300 .mu.l/min. for semi-micro and micro HPLC systems, the issue of gases dissolved in eluent (so-called air trouble) which is presently a problem is much more serious in order to maintain the accuracy of eluent delivery.
This air trouble is caused by two kinds of air; one is the air contained as bubbles (so-called air bubbles) in the eluent and the other is the one dissolved in it.
Generally, the eluent is reserved in such a container as a reagent bottle (first reservoir 1 as shown in FIG. 5). When it is drawn from the reservoir 1 directly by the pump, the reservoir and the pump are connected by a capillary tube 3 such as PTFE tube. Therefore, the eluent always passes through the capillary tube and is passed into the pump 5. When operating the pump 5, it is first checked whether or not there are air bubbles inside the capillary tube. If air bubbles are observed to be existing, they are removed from the tube by manual operation and then the eluent delivery is started. Even if no air bubble is recognized, problems may occur due to oxygen dissolved in the eluent in such a case that the eluent delivery needs to be stable over a long period of time in the area of the flow rates required for micro HPLC. The reason this may be so is because in the suction process of the eluent delivery pump the inside of the pump is in the state of reduced pressure, causing the dissolved oxygen in the eluent to become air bubbles which make the eluent delivery unstable.
For the above two problems, countermeasures are presently taken as explained in the following (1) and (2).
For air bubbles inside the capillary tube connected to the suction port of the pump, a small air bottle (air trap) is provided in front of the suction port, thereby separating the air bubbles and the liquid (eluent) so that the pump can draw the liquid component only. FIG. 6 is a block diagram to explain a compositional example of the air trap. The eluent 2 in the reservoir 1 is drawn up through the capillary tube 3 by the delivery pump 5 (not shown in FIG. 6). The air trap 4A is installed in the capillary tube 31 before the pump 5. The capillary tube 31 from the reservoir 1 is fed into the upper side of the hermetically sealed air bottle 4A with its end opened, and the other capillary tube 41 connected to the pump 5 is connected to the bottom of the air bottle. If the eluent 2 containing air bubbles is transferred into the air trap 4A, the air component (gas phase) and the liquid component (liquid phase) are separated to stay in the top and in the bottom of the air trap, respectively. The pump 5 draws only the liquid component from its bottom. The air trap 4A is also provided with a capillary tube 32 and a valve 33 in its top to take the separated air component out of it. That means, when the pump 5 draws the eluent inside the air trap 4A, the pressure in it is reduced. As the inside of the reservoir 1 is in the atmospheric pressure, the eluent 2 is sent through the capillary tubes 3 and 31 into the air trap 4A. If air exists in the eluent, the components of air and liquid are separated and stored in the upper and lower portions of the air trap 4A, respectively. Thus, the pump 5 is able to deliver the eluent alone without sucking the air. Air is gradually collected in the air trap 4A as time goes by, which is removed suitably through the capillary tube 32 by opening the valve 33.
For air bubbles existing inside the capillary tube connected to the suction port of the pump, a degassing unit (degasser) is installed prior to the suction port to separate the air component from the liquid (eluent) so that the pump can draw the liquid component only. FIG. 7 is a block diagram to explain one construction example of the degasser. The eluent in the reservoir is drawn through the capillary tube 31 by the pump, and the degasser 4B is installed in the capillary tube 31 prior to the pump 5. The degassing module is composed of a number of tubes made of such gas permeable resin film as PTFE with multi-connectors 16a and 16b connected at their both ends. While the eluent passes through the tubes, gases dissolved in it are extracted to the vacuum chamber 13, thereby avoiding the generation of air bubbles in the eluent delivery pump 5 during its suction process. However, mainly removed by this degasser is dissolved oxygen, and large air bubbles passing through the degasser. It apparently looks that the air trouble can be solved by using the air trap, but practically there are some points of problems that still are a cause for trouble.
The first problem is that it is not feasible to keep good conditions on the operation of HPLC system for continuous use over a long period of time.
When using the air trap, it is required to check an amount of air in the trap, which needs to be removed by manual operation if too much air is included. Under this kind of operational conditions, it is not possible to check all the time the amount of air which is gradually accumulated during the operation for long time, thus making it difficult to run the pump continuously. Consequently, the means of removing air bubbles by using the air trap cannot be adopted under such conditions that an automated instrument with the pump built in is operated continuously for several (ten) days.
The second problem is that the smaller the amount of eluent delivery becomes in the area of micro HPLC, the larger is the possibility of causing the air trap not to work effectively. As the aforementioned PTFE tube, in many cases, is used for the eluent delivery, air penetrates from outside into the inside of the tube through its film when the pump is stopped at night, which causes air bubbles inside the tube of the inlet side. This type of visible air in large amount is removed by the air trap. In the area of micro eluent delivery, however, it becomes a problem as an extremely fine amount of air bubbles is created inside the pump when the eluent is drawn into it. For example, at a delivery rate of several ten .mu.l/min., even air bubbles of below 1 .mu.l/min. greatly lose the stability of the eluent delivery. It is considered that small air bubbles created inside the pump are caused by temperatures of the eluent being different between in the air trap and inside the pump.
Usually, the temperature of the air trap is close to the room temperature as the air trap is installed outside the pump.
On the contrary, the temperature inside the pump is higher than a normal room temperature because heat generated by the motor and other electrical units transfers to the pump. The lower the temperature, the larger the degree of gas dissolution in liquid, and therefore, when the liquid in the air trap is drawn into the inside of the pump at higher temperature the degree of gas dissolution reduces. Air bubbles are generated by this difference in temperature. When the pump is in the suction process, its inside is at reduced pressure, which causes more air to be created. The volume of small air bubbles is relatively negligible if the volume of such a pump as a conventional one is large.
However, if the pump becomes small in its volume of 32-8 .mu.l/min. in the area of micro eluent delivery, even small air bubbles are no longer negligible.
In this connection, if no consideration is made for the change in gas dissolution degree due to the change in liquid temperature in the area of the micro eluent delivery, it is difficult to get the stable eluent delivery over a long period of time.
On the other hand, there are problems to use conventional degassers for the micro eluent delivery as they are made for conventional HPLC.
The biggest problem is that their degassing module is too large in capacity. As explained in FIG. 7, the conventional degasser removes gases dissolved at edge portions of air bubbles by passing the eluent through the inside of the degassing module composed of a number of PTFE tubes which outsides are at reduced pressure.
If the volume of this degassing module is 12 ml and if the pump delivery speed is 1-1.5 ml/min., the degassing module is most suitable in capacity.
However, it is too large if the delivery speed is in the order of 0.3-0.05 ml/min. or 0.05-0.005 ml/min. Thus, to meet the micro eluent delivery requirements, it is essential to make the capacity of degassing module small.
It is possible to make the volume of this type of conventional degassing module simply small by making the length of PTFE tubes (typically 2,500 mm) short. However, it deteriorates the replacement efficiency of the eluent inside the degassing module, resulting in a lower efficiency of degassing.
The reason for this is because as PTFE tubes are made shorter the difference of flow resistance created when the eluent passes in the tubes becomes larger, thereby causing the eluent replacement efficiency to get worse. If this is simply explained, it is because the eluent in PTFE tubes of a small resistance passes in a short time.
Many of those who use solvent delivery pumps seem to think that air bubbles can effectively be removed by using a degasser. But, it is wrong. Probably, the term degasser may give some image that all air troubles can be solved if it is used.
The purpose of the "degasser" is to remove gaseous components dissolved in the eluent, but not to remove air bubbles themselves.
For example, the amount of oxygen dissolved in water is 8-9 ppm in maximum, which can be reduced to more or less 1 ppm by the use of the degasser. Thus, it may be considered that the degasser can reduce approx. 8 ppm in oxygen dissolution. Suppose that the pump is delivering the eluent at a rate of 1 ml/min., the volume of air (oxygen) corresponding to the change of the dissolution, which is removed by the degasser, is 5.6 .mu.l.
The volume of this, if applied to air bubbles, represents extremely small air bubbles, and large ones that are clearly visible cannot be removed by the degasser.
Even if the capacity of the degasser is increased five times, the aforementioned value becomes 28 .mu.l/min. With a degasser capacity made 1/10, it is 2.8 .mu.l/min. Thus, if air of more than these volumes comes into the degasser, it passes through the degasser.
It is understood from the above explanations that it is difficult to maintain the stable eluent delivery over a long period of time in the area of the micro eluent delivery even if the conventional degasser and the air trap are used simultaneously. Therefore, it is needless to say that a new type of degasser (micro degasser) is required to meet the requirements of the micro eluent delivery.
The new micro degasser has been invented by Mr. Toshinori Saitoh, the inventor of this invention, which initial patent application was filed on Nov. 11, 1997 in Japan, followed by a U.S. patent application with the priority claim filed on Nov. 4, 1998 as Ser. No. 09/186,870, now U.S. Pat. No. 5,980,742, granted Nov. 9, 1999.
The micro degasser is provided with a flat film degassing module in small volume, thereby allowing better efficiency in eluent replacement as well as in degassing than any conventional degassers.
FIG. 8 is a cross sectional view of said micro degasser. In this figure, the degasser 4C is provided with a thin flat space to pass the eluent, which is formed by at least two PTFE film sheets instead of using PTFE tubes which is shown in FIG. 7.
The eluent from a reservoir which is not shown in the figure flow into a degassing module 16' through a capillary tube 31 and a connector 104. While the eluent flows and passes between the PTFE film sheets, the air components dissolved in the eluent are extracted to the inside of a vacuum chamber 13, allowing the eluent with air removed to be delivered by a pump 5 through a connector 105 and a capillary tube 41. Further explanations are not believed necessary as the other components are the same as in FIG. 7.
For details on this type of liquid chromatography and degassing unit, refer to U.S. Pat. No. 5,472,598.
It has been attempted to change the eluent delivery pump system as a means to solve troubles caused by air bubbles. Solvent delivery pumps for conventional HPLC are classified into two systems as shown in FIGS. 9(a) and 9(b). Those illustrations are to explain the types of conventional delivery pump systems and their relations with air troubles. FIG. 9(a) shows a parallel delivery pump system and FIG. 9(b) a series delivery pump system, respectively, in which 50A and 50B represent pump plungers.
The parallel delivery pump system in FIG. 9(a) is most popular, wherein the two plungers 50A and 50B are connected in parallel. The eluent is drawn respectively into the plungers 50A and 50B, and is then sent out alternately from them to keep the eluent delivery continuously, as shown by the arrow marks.
In the series delivery pump system as shown in FIG. 9(b), on the other hand, those two plungers 50A and 50B are connected in series, wherein the eluent is drawn into the first step plunger 50A as shown by the arrow mark. The half amount of the eluent sent out from the plunger 50A is drawn by the next step plunger 50B. While the plunger 50A draws the eluent, the plunger 50B sends it out so that by repeating these operations the eluent can be continuously delivered.
Among the above mentioned two pump systems, air bubbles sucked by the pump are removed more readily in the series delivery system of FIG. 9(b) than in the parallel delivery system.
One of the reasons for easier air bubble removal lies in the volume of the plunger. In the series pump system of FIG. 9(b) the volume of the plunger 50A used is two times that of the plunger 50B, which is also two times that of the both plungers used in the parallel pump system of FIG. 9(a).
The use of such big volume plunger as in the series pump system provides one of the reasons for the series pump system with bigger capability to remove air bubbles than the parallel pump system.
The second reason why the series pump system of FIG. 9(b) is better in air bubble removal is in the configuration of the plungers. In this system the two plungers 50A and 50B are configured in series, wherein while the first step plunger 50A sends the eluent out the second step plunger 50B draws it in. Consequently, these operations allow air bubbles to be removed more readily than in the parallel pump system.
As such, it is possible to make air bubbles readily removable by modifying the pump system. However, the eluent delivery rate becomes unstable while air bubbles are existing inside the pump.
Another difference between the pump systems of FIG. 9(a) and FIG. 9(b) is that the time of the delivery or rate is unstable when air bubbles are present as the pump is shorter in the series system of FIG. 9(b) than in the parallel system of FIG. 9(a).
In this connection, it does not mean that the air trouble can be thoroughly solved by modifying the conventional pump system, but it should be considered that the air trouble becomes less of a problem in the series pump system than in the parallel pump system and that removing the air bubbles is fundamentally difficult.